Bacterial resistance to antimicrobial agents is a significant threat to the efficacy of antibiotic therapy. The ?-lactam antibiotics, such as the penicillins and cephalosporins, are among the most often used antimicrobials. The most common source of resistance to ?-lactam antibiotics is the production of ?-lactamases, which cleave the drugs and render them ineffective. ?-lactamases have been grouped into four classes based on primary sequence homology. Classes A, C and D are active-site serine enzymes that catalyze the hydrolysis of ?-lactam antibiotics via a serine-bound acyl-enzyme intermediate. Class B enzymes require zinc for activity and catalysis does not proceed via a covalent intermediate. Because of the wide range of substrate specificities of these enzymes, virtually all ?-lactam antibiotics are susceptible to hydrolysis. Clearly, the design of new antibiotics that escape hydrolysis by the growing collection of ?-lactamase activities will be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of each class of ?-lactamase. The goal of this work is to understand how the amino acid sequence determines the structure, activity and evolution of class A ?-lactamases. The TEM-1 and CTX-M family of class A enzymes are an important cause of resistance to ?-lactam antibiotics worldwide. In a previous funding period in vitro mutagenesis was used to randomize the entire coding sequence of the class A TEM-1 ?-lactamase. The sets of random mutants were used to identify residues that are critical for the folding, stability and substrate specificity of the enzyme. The recent advent and application of ultra-high throughput sequencing technology has allowed the scope of this approach to be vastly extended to accurately determine the sequence requirements for hydrolysis of multiple classes of ?-lactam antibiotics for every residue position in TEM-1 ?-lactamase. In addition, preliminary studies suggest that amino acid substitutions which increase the stability of TEM-1 ?-lactamase play an important role in the evolution of TEM-1-mediated resistance to extended-spectrum ?-lactam antibiotics and ?-lactamase inhibitors. These studies will be continued to understand how these changes influence the stability and activity of evolved enzyme variants. Finally, the CTX-M family of ?-lactamases exhibits a high degree of sequence variation and it is hypothesized that many of the amino acid substitutions observed alter the stability or substrate specificity of the enzymes. Experiments are proposed to test this hypothesis by examining the role of a selected set of amino acid substitutions on the structure, activity and evolution of CTX-M enzymes.